Parathyroid hormone is an 84 amino acid peptide secreted by the parathyroid gland. It's physiological role is maintaining serum calcium, and bone remodelling (Dempstor D. W. et al., Endocrine Reviews, 1993, 14, 690-709). The remodelling of bone is typically a 3 to 6 months process wherein there is a coupling between bone resorption and bone formation. Estrogens, vitamin D, bisphosphonates are known to inhibit bone resorption where as rhPTH (1-34), an anabolic agent, increases bone mass. Osteoporosis is a disease that makes a bone susceptible to fracture and rhPTH (1-34) is a peptide drug that has revolutionized treatment of osteoporosis. RhPTH (1-34) when given alone stimulates bone mass formation and causes a net increase in bone mass in each remodeling cycle, wherein bone mineral density is increased by 10% per year typically in lumbar spine area.
Parathyroid hormone (PTH) is secreted by the parathyroid glands in response to a decrease in plasma calcium concentrations and has several effects that act to restore normal calcium levels. It has been shown that PTH has both anabolic and catabolic effects on the skeleton. Persistent elevation of PTH causes increased bone resorption, whereas intermittently administered PTH results in enhanced bone formation (Canalis, E., Hock, J. M., and Raisz, L. G. (1994) in The Parathyroidds: Basic and Clinical Concepts, ed by Bilezikian, J. P., Marcus, R., and Levine, M., Raven Press Ltd., NY) although the cellular mechanism of this dual effects is not clear yet.
PTH is biosynthesized as a 115-amino acid precursor, preproparathyroid hormone (preproPTH). Although the anabolic actions of PTH(1-84) have been known, the actual structural requirement for full biological activity lies in the residues 1 through 34 in the N-terminal of the molecule (PNAS 68, 63-67, 1971; Endocrinology 93, 1349-1353, 1973).
PTH is particularly sensitive to oxidation especially at its methionine residues, Met8 and Met18 and it requires the intact N-terminal sequence for preserving its bioactivity. Frelinger, A. L., et al, (J. Biol. Chem., (1984) 259 (9), 5507-5513) developed a method to separate the methionine oxidized PTH from the native molecule and have shown that the potency of the oxidized molecule is dramatically less than the native PTH. For its comparable bioactivity to the full length PTH, PTH (1-34) requires both the N & C-terminal helical conformation as shown by Jin. L. et al, (J Biol. Chem., (2000) 275 (35), 27238-44. Their model proposes a receptor binding pocket for the N-terminus of PTH(1-34) and a hydrophobic interface with the receptor for the C-terminus of PTH(1-34). Pellegrini M, et al., J. Biol. Chem. (1998) 273 (17), 10420-427 elucidates the high resolution structures of hPTH(1-34) in aqueous solution investigating the effect of pH and salt concentration on secondary and tertiary structures by CD and NMR. The helix content of PTH(1-34), based on CD spectra, increases in the presence of acidic buffer as against benign water. The NMR studies confirm the presence of helical structure localised to the N- & C-terminal part of hPTH(1-34). The molecule outside the context of receptor interaction is far too flexible to prefer any definite secondary or tertiary fold, wherein experimental distance restraint at 0.06 A° gives a flexible rod shape without receptor interaction and “U” shape to the protein while interacting with the receptor. It has been shown that intermittent exposure of the ligand to its receptor has a preferred anabolic effect.
Cloning and expression of a therapeutic protein poses an early challenge in having an appropriate genetic material. Oligonucleotide primers used for cloning should not be degenerate, should have comparative tm and most land at an unique location in the gene of interest. RNA transcript of PTH (1-84) gene is found to be positive in liver, kidney, brain, and placental human tissues. Gene specific primers when used with polymerase chain reaction are suitable in an RT-PCR reaction to clone in the cDNA fragment of specific sequence. It is advisable to fully sequence the insert prior to recloning of the gene. It is well known in the art about many Escherichia coli expression vectors available for suitably ligating a cloned fragment, e.g. HB101, JM109, BL21(DE3), TOP10 for high level heterologous expression of recombinant proteins. Primer pairs used to clone in the tag nucleotide sequence can be engineered to contain restriction sites for sequential cloning steps which contain gene of interest with protease cut site and affinity tag.
U.S. Pat. No. 5,496,801 teaches the hPTH preparations that exhibit storage stability in terms of the hormone composition and stability. U.S. Pat. No. 5,496,801 advocates the lyophilization of hPTH (1-84) with mannitol as a cryoprotectant and a non-volatile citrate buffer in the pH range of 3.5 to 6.5 to yield a stabilized ready to use liquid formulation for parenteral administration.
U.S. Pat. No. 4,086,196 discloses for the first time that all fragments of PTH greater than 1-27 hPTH and (Ala1)-hPTH (1-27) have useful biological properties. U.S. Pat. No. 4,086,196 inherently claims hPTH (1-X) and Ala1hPTH (1-X) wherein X is Ser or Ala.
U.S. Pat. No. 4,086,196 also teaches the synthesis of hPTH (1-34) by Solid Phase Peptide Synthesis (SPPS) by the methods well known in the art. U.S. Pat. No. 4,086,196 also discloses the recovery of the bioactive hPTH (1-34) by gel filtration followed by ion exchange chromatography on Whatman-CM-52 and elution carried out following linear gradient using ammonium ions as counter ions. The chief limitations of gel filtration chromatography (GFC) are slow separation and lower peak resolution attributed to factors inclusive of improper matrix selection, column length, high flow rate and large dead spaces entrapped within the column. However, GFC is indeed an attractive option for desalting and buffer exchange for peptide and protein purification. Also chemical synthesis often involves high risk and cost, and although production via recombinant genetic technology has been expected to replace this process, the yield has so far been insufficient. Moreover, the synthesis thereof requires utilizing techniques which require a high level of skill and expertise. Accordingly, the production of hPTH using recombinant DNA techniques is desirable
Because of its ease of cultivation, low cost and high production potential, Escherichia coli is a preferred host to express and purify pharmaceutically important proteins. However in the case of hPTH due to inherent instability associated with direct expression of the protein (Morelle et al 1988, Biochim. Biophys. Acta 950, 459-462.) a fusion protein strategy has been adopted. Fusion partners commonly used include β-galactosidase, cro-β-galactosidase, hGH, Trx and phospho ribulokinase (Suzuki, Y et al, Appl. Env. Micro 1998, 64, 526-529, Wingender E et al, J. Biol. Chem 264, 4367-4373, Gardella T J et al, J. Biol. Chem 26, 15854-15859, Xiang Yang Fu et al, Biotechnol. Prog 2005, 21, 1429-1435, WO/1999/005277, C12N15/62) for expression and downstream purification of this protein. Also the number of amino acids that were used from β-galactosidase as fusion partner were different for different proteins. In case of insulin and proinsulin (Shen, S-H. (1984), Proc. Natl. Acad. Sci. USA 81: 4627-4631, Guo, L. (1984), Gene 29: 251-25) the β-Gal fusion partner was much longer than PTH on the same promoter back (T7 and lac promoter, Massayuki, Y et al. (1997), U.S. Pat. No. 5,670,340). Much literature reports the expression of fusion protein as insoluble aggregates by subjecting to a series of denaturation and refolding steps. In cases where β-galactosidase have been used as the fusion partner for PTH(1-34), final yields ranging from 20 mg/L to 500 mg/L have been reported using Isopropyl beta galactoside (IPTG) as the inducer at 1 mM concentration. However, the use of IPTG for the large-scale production of recombinant proteins is undesirable because of its high cost and toxicity (Donovan et al., 1996; Figge et al., 1988; Gombert and Kilikian, 1998; Ksinski et al., 1992). Oldenburg K. R., et al (Protein Exp. Purification 5(3), 278-284, (1994)) describes a method for high-level expression of rPTH(1-34) in E. coli, with polyhistidine leader peptide and eight copies of PTH gene. Oldenburg K. R., et al also teaches the fusion protein capture by Ni chelation chromatography followed by a cyanogen bromide (CNBr) cleavage and a purification by RP-HPLC with a final yield of 300 mg/L of highly purified biologically active hPTH(1-34). Use of cyanogen bromide poses an environmental threat in terms of safe handling Wand disposal which severely restricts the use.
EP 794255 disclose purification of rhPTH (1-34) using Kex2 cutting from its chimera. Suzuki Y, et al., (Applied and Environmental Microbiology 64(2), 526-529 (1998)) obtained 0.5 g of >99% pure hPTH (1-34) from one liter of Escherichia coli culture using different lengths of β-galactosidase linker along with His-tag fusion partner. Suzuki Y., et al., disclose the isolation of the inclusion bodies, solublisation in 8M urea, dilution to have a urea concentration of 3M followed by Kex2 digestion followed by intermediate purification by ion exchange chromatography, and final polishing by two steps of reverse phase chromatography. Suzuki Y. et al., teaches that, Kex2 a secretory type Kex2 protease from yeast used for enzymatic cleavage is significantly affected by the 3M Urea concentration required for maintaining the fusion protein in the soluble form, even addition of 2.5 mM CaCl2 to suppress the inactivation lead to precipitation of the fusion protein which resulted in use of molar ratio of 1:2000 for enzyme to substrate. Suzuki Y. et al., thus though reports high yield of the target protein but Kex2 usage still accounts for too large a proportion of the primary cost of the production process. Suzuki Y. et al., also explicitly teaches the use of 97,117 and 139 amino acid fragments of β-galactosidase as fusion partner spaced by linker having SVKKR (SEQ ID NO:9) as the cleavage site for Kex2 accounts for high yield of the fusion protein.
Jin. L., et al, J Biol. Chem. (2000), 275(35), 27238-27244) discloses a purification process for LY 333334 molecule rhPTH(1-34) by solublisation in 7M Urea and capture on a reverse phase column, followed by a FF SP cation exchange column purification using a NaCl gradient subsequently followed by a RP-HPLC method wherein the purified material in 20 mM Glycine buffer pH 9 is freeze dried. This is a pro-solvent method and may be hazardous at manufacturing scale. Fu, Xiang-Yang et al, (Biotechnology Progress (2005), 21(5), 1429-1435) teaches a method to purify a thioredoxin fusion of PTH(1-34) from BL21(DE3) cells by use of Triton-X 100 and heat denaturation induced partial purification by precipitation. The heating is done at 80° C. for 15 minutes. The method treats the protein at high temperature which is generally undesirable for proteins and peptides. CN 1417231 describes a process for recovery of recombinant PTH(1-34) by fermentation, followed by inclusion body purification, renaturation, thrombin digestion and purification over cation exchange chromatography. CN 1424325 describes a GST fusion PTH(1-34) peptide (with GSP as the cleavage site) digested with thrombin, purification on chymotrypsin affinity column, digestion with proline endopeptidase and further chromatographic purification. The process is cumbersome and use of two proteases adds to the cost of the purification process limiting the commercial exploitation of the same. Biochem. Biophys. Res. Commun., 166, 50-60 (1990) documents a cDNA approach for the synthesis of hPTH.
Chen, J. Y. et al, ((2004), 40 (1), 58-65) discloses PTH(1-34) purification, by expression of PTH (1-34) as a chimera with cellulose binding domain, cleaving PTH(1-34) by Factor Xa, purifying with cellulose resin and RP-HPLC to yield 3 mg/L which is a very low yielding process with no commercial viability. GST fusion technology for PTH (1-34) production is also well known in the art. Gram Hermann et al, (Bio/Technology (1994), 12(10), 1017-23) describes a method for purification of PTH(1-34) using dipeptidyl peptidase IV. Wingender E., et al, (J Biol. Chem. (1989), 264(8), 4367-4373) expressed PTH in E. coli as cro-β-galactosidase-hPTH fusion protein. An yield of about 250 mg of fusion protein was obtained from 1 L of culture which they solublised in Urea and further, an acid treatment was used to release PTH. Acid cleavage conditions are plagued by limitations due to formation of deamidated or oxidised related protein impurities which are difficult to separate in some proteins and moreover acidic pH poses harsh conditions detrimental to protein bioactivity.
EP 0483509B1 relates to a codon optimized synthetic gene producing hPTH corresponding to the amino acid sequence of hPTH, DNA containing it, a host cell transformed by the DNA and a method for producing hPTH using the transformant in E. coli with IPTG induction. EP083509B1 discloses the purification of the PTH expressed by RP-HPLC. Use of organic solvents as eluants in RP-HPLC may harm the protein which poses scalability problem. U.S. Pat. No. 5,208,041 teaches production of essentially pure hPTH characterized by single peak migration when analyzed by capillary electrophoresis at 214 um, and by an EC50 as determined in the UMR 106-based adenylate cyclase assay of not more than 2 nM, by purifying the crude hPTH by RP-HPLC with a cationic ion-pairing agent as triethylamine phosphate. U.S. Pat. No. 5,208,041 also discloses subjecting the hPTH obtained either from mammalian tissue, from microbial sources of PTH or from synthetic sources to at least one column fractionation step prior to RP-HPLC. U.S. Pat. No. 5,208,041 exemplifies the purification process by subjecting the whole broth to pH adjustment from 4.0 to 8.0 with glacial acetic acid, clarification by centrifugation, followed by loading on to an ion exchange chromatography column of S-Sepharose, further subjecting the eluate to an intermediate purification step by loading on to HIC column of Phenyl Sepharose and finally purifying by RP-HPLC using C18 column with triethylamine phosphate as the ion pairing agent. U.S. Pat. No. 5,208,041 cites the detection by capillary electrophoresis of 4 previously undetected minor peaks eluting ahead of PTH peak and some trailing peaks which were not detected by using trifluoroacetic acid (TFA) or heptafluorobutyric acid (HFBA) as ion-pairing agent thus leading to the recovery of essentially pure hPTH, U.S. Pat. No. 5,208,041 does not in any way teaches the use of HIC as final purification step to obtain a purified hPTH (1-34) with ≧99% purity which is the focus of the present invention. U.S. Pat. No. 5,457,047 relates to DNA sequences coding for PTH variants, expression vectors, bacterial hosts, uses and therapeutic compositions. U.S. Pat. No. 5,457,047 discloses hPTH purification by means of CM-cellulose in batch mode followed by RP-HPLC from cro-β-galactosidase-hPTH fusion protein.
U.S. Pat. No. 6,590,081 teaches the synthesis of pure crystalline form of teriparatide, and methods of preparation and purification of the fragmented PTH. U.S. Pat. No. 6,590,081 explicitly teaches the advantage of crystalline form of the hormone to be product purity and storage stability. Also to create more potent and orally available analogs of PTH, detailed structural information on the peptide should aid in characterizing the molecule interactions between the ligand and the receptor. U.S. Pat. No. 6,590,081 also discloses that the crystalline PTH may also be formulated into other compositions such as for example, tablets, capsules or suppositories, as the same is easily dissolved in sterile solution in vials. U.S. Pat. No. 6,590,081 claims the cubic, hexagonal and plate like crystals of hPTH(1-34) and process for purifying the PTH to obtain the same.
Liu, Q., et al., in Protein Expr Purif., 2007 August, 54(2): 212-9, disclose a large scale preparation process of hPTH (1-84) from E. coli using a soluble fusion protein strategy, by constructing a codon-optimized synthetic gene encoding hPTH(1-84) and cloning the same in pET32a (+) vector, expressing in E. coli BL21 (DE3) cells as soluble His(6)-thioredoxin-hPTH(1-84) fusion protein. Liu, Q., et al., follows a sequel of purification steps of capture by immobilized metal affinity chromatography, followed by enterokinase cleavage and terminally subjecting the same to size exclusion chromatography with a quantified yield of 300 mg/L with a purity of 99% after harvesting the soluble fusion protein.
Orthogonal process design is the foundation of well-controlled purification procedures (Gagnon P. The secrets of Orthogonal Process Development. Validated Biosystems, 2006:www.validated.com/revalbio/pdffiles/orthopd.pdf). The idea is that combining the steps with the greatest complementarity should provide the best overall purification. The strongest embodiment of the concept is usually achieved when respective steps are based on distinct separation mechanisms. A two-step process that contains one fractionation step based on product size and another on product charge would be considered orthogonal; similarly, a process with one step based on product charge and another on hydrophobicity would also be orthogonal. An important feature of orthogonal process design is that the purification capability of any one step is measurable only within the context of its potential partner.
The present invention comprises an orthogonal process for purification of rhPTH (1-34) coupling cation exchange chromatography to HIC to achieve a purity of ≧99% with good yield.
The large-scale production of therapeutic proteins faces several challenges, such as short development timelines, cost considerations, and ever increasing quality requirements. State-of-art isolation and purification technologies, specifically orthogonal separation principles, are of enormous importance to speed up development, shorten processing times, and cut the production costs. Preparative chromatography has advanced significantly with regard to matrix stability or availability of selectivities, and provides the premier technology in biomolecule purification to purities above 95%. Hydrophobic interaction chromatography (HIC) has gained popularity in recent years since it offers orthogonal separation power to widely used purification techniques based on ionic interactions. HIC is often an excellent choice subsequent to ion exchange chromatography in a protein purification procedure. Both techniques have an extremely broad applicability and are canonical approaches with respect to one another (i.e. separation according to hydrophobicity and charge respectively). Furthermore, material eluted with a salt gradient in an ion exchange separation requires a minimum of sample treatment. On the other hand, while exerting similar selectivity, HIC is less denaturing compared to reverse phase chromatography, using more hydrophobic ligands and organic solvents. At an industrial scale, explosion-proof production suites are required for the handling of the toxic organic solvents, the disposal of large quantities of which is costly. Hence an orthogonal approach of coupling ion exchange chromatography followed by HIC is a cost-effective, environmentally benign platform technology for large scale production of recombinant fusion proteins.
Above cited prior art neither explicitly teach the orthogonal approach of purification of hPTH(1-34) by coupling cation exchange chromatography as an intermediate purification step to HIC as the final purification strategy, nor, reports the use of novel lactose induction with good yield of hPTH (1-34), which is the prime focus of the present invention.
The present invention has an unique, novel feature of use of an orthogonal approach of two step purification process of cation exchange chromatography optionally followed by preparative chromatography selected from HIC or RP-HPLC. The present invention discloses a simple, cost-effective, environmentally benign method of producing high purity hPTH (1-34). Other unique feature of the present invention is lactose induction for a fed batch production strategy for optimized expression of hPTH (1-34) in prokaryotic host. Another feature of the present invention is construction of a chimeric fusion protein comprising of a fusion partner consisting of 41 amino acids of Escherichia coli β-galactosidase (LacZ) gene, an endopeptidase cleavage site, rhPTH gene fragment wherein the fusion partner, the β-galactosidase gene is chosen being a protein native to Escherichia coli, high % GC content, corresponding peptide secondary structure, the secondary structure of ribonucleotide translating the same, and the pI of the fusion fragment as an aid facilitating downstream processing.